What this is
- This research investigates the therapeutic targeting of hexokinase 2 (HK2) in cancers that express only HK2 while lacking hexokinase 1 (HK1).
- HK2 is a key enzyme in glycolysis, which is often upregulated in cancer cells to meet their energy demands.
- The study identifies a combination therapy involving HK2 inhibition and mitochondrial complex I inhibition as potentially effective for HK1-null, HK2-positive cancers.
Essence
- HK1-null, HK2-positive cancers can be targeted using a combination therapy that inhibits HK2 and mitochondrial complex I, leading to reduced energy production and potential cancer cell death.
Key takeaways
- HK1HK2cancers are sensitive to HK2 silencing, unlike HK1HK2cancers, which do not respond. This distinction is crucial for developing targeted therapies.
- The combination of diphenyleneiodonium (DPI), a mitochondrial complex I inhibitor, and HK2 inhibition achieved in HK1HK2liver cancer cells.
- Adding perhexiline, a fatty acid oxidation inhibitor, further sensitized HK1HK2liver cancer cells to the combination therapy, suggesting a multi-targeted approach may enhance treatment efficacy.
Caveats
- The study primarily focuses on liver cancer cell lines, which may limit the generalizability of the findings to other cancer types.
- While promising, the combination therapy's effectiveness in clinical settings requires further validation in human trials.
Definitions
- Synthetic lethality: A situation where two non-lethal mutations lead to cell death when combined, providing a potential therapeutic strategy.
AI simplified
Background
Precision medicine depends on the identification of a unique molecular cancer subtype that may exist across tumors with different origins. The recent Food and Drug Administration (FDA) approval of Keytruda for treatment of a wide range of advanced cancers with the common biomarkers microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) illustrates this concept of phenotype/genotype commonality, rather than tissue of origin, for a common therapeutic approach to subclasses of cancers from tissues of varying origin [1]. Larotrectinib, which targets the rare tropomyosin receptor kinase (TRK) fusion mutation, is another example of precision cancer medicine; larotrectinib has demonstrated efficacy in cancers from different tissues that share TRK fusion mutations [2]. FDA approval is currently being sought for the larotrectinib treatment of adult and pediatric TRK fusion cancers, based on molecular makeup rather than the tissue of origin [3].
Most cancers increase glycolysis (the “Warburg effect”), a metabolic event postulated to ensure sufficient supplies of energy (ATP), reducing equivalents (NADPH), and/or biochemical building blocks for cell growth and proliferation [4]. However, because of the conserved nature of the glycolytic pathway in normal tissues, global systemic inhibition of glycolysis results in adverse effects that make this approach of limited value; selective inhibition of cancer-driven glycolysis will be required for clinical cancer therapy.
The hexokinase (HK) enzymes, encoded by four genes (HK1/2/3/4), catalyze glucose phosphorylation, the first enzymatic step in glycolysis [5]. Most adult tissues use HK1 for glycolysis. HK3 is inhibited by physiological concentrations of glucose. HK4, also known as glucokinase (GCK), is expressed in hepatocytes, pancreatic β-cells, and glucose-sensing neurons. HK2 is expressed primarily in embryonic tissues and in adult muscle and adipose tissues. In addition, HK2 is expressed in a wide range of cancers [6–9], from tissues that normally express only HK1. HK2 gene deletion in adult mice does not significantly affect normal tissues [6]. Consequently, selective HK2 inhibitors have recently been developed [10], under the assumption that targeted HK2 inhibition will reduce progression of HK2-positive cancers and have minimal adverse effects. However, most previous studies on HK2 in cancer did not examine the contribution of HK1 to cancer glycolysis [6–9, 11]. Cancer subtypes with differential HK1/HK2 expression have not been characterized.
In addition to enhanced glycolysis, other modes of energy generation are utilized to support biological processes in cancer cells; these alternative energy-generating sources include oxidative phosphorylation and fatty acid oxidation [12, 13]. The availability and use of multi-source energy generation alternatives suggests both the flexibility of cancer cells in reprogramming their required energy generation under metabolic stress and their abilities to escape from alternative modes of energy generation blockade using monotherapies. Combination therapies that target compensatory energy metabolism pathways in cancer cells, but are tolerated by normal tissues, have not yet achieved clinical success, due to the conserved nature of energy metabolism shared by most cancer and normal cells.
We have developed a combination therapy specifically effective in cancers with the HK1−HK2+ molecular signature, a subset of cancers which originate from a wide variety of tissues. Using an HK1−HK2+ subpopulation of liver cancers as an example, we developed a synergistic combination of HK2 inhibition and partial inhibition of mitochondrial complex I and fatty acid oxidation to achieve synthetic lethality of these HK1−HK2+ tumors. However, like HK1+ cells/tissues, HK1+HK2+ cancers are not susceptible to this therapy, despite the reduction in their HK2-driven glycolytic activity. Our findings warrant optimization of the triple-combination therapy as a precision medicine for further clinical development to treat HK1−HK2+ cancers, regardless of their tissues of origin.
Methods
Cell lines and tissue samples
Hep3B, HepG2, H460, H1299, HT29, HCT116, SW620, and Caco2 cells were from the American Type Culture Collection (ATCC). Huh7, JHH7, JHH5, and MDAMB468 cells were provided by Drs. Dennis Slamon and Richard Finn (UCLA). SUM159, MDAMB231, and HCC1937 cells were provided by Dr. Heather Christofk (UCLA). A549, SKOV3, OVCAR5, and HEY cells were provided by Dr. Caius Radu (UCLA). Hs766T, MiaPaca2, and Panc1 were provided by Dr. Timothy Donahue (UCLA). LNCaP, DU145, and PC3 cells were provided by Dr. Hong Wu (UCLA). 786-0 cells were provided by Dr. Allan Pantuck (UCLA). All cells used for the experiments were between passages 3 and 20. Cell lines were routinely authenticated based on morphology, growth characteristics, and HK expression profiles according to the CCLE gene expression dataset. All cells were maintained in RPMI1640 + 10% FBS, at 37 °C in 5% CO2/95% air. Cells were routinely checked for Mycoplasma contamination by using MycoAlert (Lonza). Frozen human liver and liver cancer samples were provided by the UCLA Translational Pathology Core Laboratory.
High-throughput screen (HTS) for compounds synergistic with HK2 knockdown in cell growth inhibition
In the primary HTS screening, libraries of 3205 drug-like small molecules and 119 FDA-approved oncology drugs were screened for their ability to inhibit the growth of Hep3B/shHK2DOX cells in the presence of DOX. Hep3B/shHK2DOX cells were pretreated with DOX for 48 h, then seeded in 384-well plates with 700 cells per well, and treated with DOX and individual library members at 10 μM for 72 h. Relative numbers of viable cells in response to different treatments were determined by the CellTiter-Glo assay (Promega). Compounds with z score < − 3 were selected for subsequent secondary screening. In the secondary screening, Hep3B/shHK2DOX cells with or without 48-h DOX pretreatment, were treated subsequently with the selected compounds in dose response curves (DRCs 10, 2.5, 0.625, 0.156, 0.039, 0.010, 0.0024, and 0.0006 μM) for 72 h. Relative numbers of viable cells were determined by the alamarBlue assay (Invitrogen).
Media metabolite measurement
Medium was collected from culture plates and analyzed for glucose, lactate, and glutamine concentrations using a Biomedical Bioprofile Analyzer (Nova Biomedical). Cells seeded in 6-well plates received treatments described in the “” section and the figure legends. Twenty-four hours before the analysis, the media were refreshed. Medium added to wells with no cells was used as a blank control. After 24-h incubation, 1 ml of medium was collected from each sample and the blank control, and media samples were analyzed in the Bioprofile Analyzer. Values were normalized to cell number and time intervals. DPI was purchased from Cayman Chemical (#81050). PER was purchased from Cayman Chemical (#16982). FDG was purchased from Omicron Biochemicals Inc. (#GLC-010). Results
In vivo assessment of treatment efficacy and safety
Nu/nu mice (Jackson Laboratory) were used for in vivo efficacy and safety studies. Mice were fed ad libitum and kept in air-conditioned rooms at 20 ± 2 °C with a 12-h light-dark period. Animal care and manipulation were in accordance with the Guidelines for the Care and Use of Laboratory Animals. Cells (Hep3B, 5 × 106; Huh7, 2 × 106; HepG2, 2 × 106; H460, 1 × 106) in 100 μl RPMI/matrigel (1:1) were implanted subcutaneously in nu/nu mice under aseptic conditions. Body weight was measured biweekly. The body condition score (BCS) was also used to evaluate animal health. Tumor growth was assessed by biweekly measurement of tumor diameters with a vernier caliper. Tumor volume (mm3) = D × d2/2, where D and d are the longest and shortest diameters, respectively. After subcutaneous tumor cell inoculation, tumors were allowed to grow to a volume of 200 mm3. We chose tumors of this size because, in our experience, tumor xenografts of smaller sizes may regress. Mice were then randomly assigned into groups as indicated in the “Results” section and the figure legends. DOX was given in the diet (625 mg DOX per kg diet, daily DOX 1.6–2.7 mg in 3–5 g diet per mouse). DPI (#D491550, Toronto Research Chemicals) and PER were dissolved in DMSO as stock solutions at 20 and 300 mg/ml, respectively, diluted in 5% (w/v) hydroxyl-propyl-beta-cyclodextrin (Sigma) as injection solutions, and given daily by intraperitoneal (i.p.) injection at 2 and 30 mg/kg, respectively.
Statistics
Student’s t-test was used for statistical analysis. P values were determined by Prism 5 (GraphPad Software, Inc.). Differences were considered statistically significant at P < 0.05.
Study approval
All reported animal studies were approved by the UCLA Chancellor’s Animal Research Committee (ARC).
Results
The conversion of liver to liver cancer is accompanied by extinction of HK4 expression and tumor expression of either HK2 alone or HK1 and HK2
Because tumor biopsies are likely to have normal tissue contamination, it is not possible to evaluate tumors in the TCGA collection from other tissues of origins for tumor HK1 and HK2 expression; the presence of HK1 derived from most normal tissues would distort the estimate of HK1 tumor-derived levels in tumor biopsies. In contrast, contamination with normal liver, which does not express either HK1 or HK2, would not affect the evaluation of HK1 and HK2 expression in liver tumor samples.
Subsets of cancers from a variety of tissues of origin are HK1HK2.HK1, HK2, and HK4 protein levels in organs. Organs were collected from two C57BL/6 male mice.HK2 expression is upregulated in clinical liver cancer samples relative to normal liver. The TCGA_Liver dataset was obtained from OASIS (), and the Roessler_Liver dataset was obtained from Oncomine (). Numbers after sample names indicate sample size. Bar: mean value.HK1 and HK2 mRNA expression profiles in the TCGA_Liver Cancer dataset. Data were obtained from the cBioPortal (). RSEM RNA-Seq by Expectation Maximization.Identification of HK1HK2cancer cell lines present in the Cancer Cell Line Encyclopedia (CCLE) collection (). Upper panel, HK1 and HK2 mRNA expression of 935 cell lines with RNASeq data in the CCLE dataset. Each data point represents one cell line. The box indicates “HK1HK2” cell lines, defined as HK2 > 1 TPM and HK1 < 10 TPM. TPM transcripts per million. Lower panel, distribution of the CCLE HK1HK2cancer cell lines in a broad spectrum of cancers of different tissues of origin.Hexokinase isoform expression in four human samples and seven human liver cancer cell lines − + − + − + − + a b c d e http://oasis-genomics.org http://www.oncimine.com http://www.cbioportal.org http://www.cbioportal.org
Tumors of multiple origin contain HK1HK2and HK1HK2subsets − + + +
To confirm and extend the observations regarding the presence of HK1−HK2+ liver cancers and to determine whether HK1−HK2+ subsets exist in cancers from other tissues of origin, we examined the frequencies of HK1−HK2+ and HK1+HK2+ subsets in established cancer cell lines of the Cancer Cell Line Encyclopedia (CCLE) collection [16]. Subsets of cancer cell lines exhibiting the HK1−HK2+ molecular characteristic are present, at varying frequencies, across a broad spectrum of cancer types from a wide variety of tissues of origin (Fig. 1d).
We chose to use liver cancer cell lines to investigate HK2 as a therapeutic target because of (i) the substantial contributions of both HK1−HK2+ and HK1+HK2+ tumor cell lines to the liver CCLE population, (ii) the lack of effective treatment for liver cancers, and (iii) the prediction of liver cancer as the third most lethal cancer in the USA in the next decade [17]. To confirm and extend these observations, we evaluated the expression of HK1 and HK2 in human liver samples and in a collection of liver cancer cell lines (Fig. 1e). At the protein level, all established human liver cancer cell lines we examined express HK2 and lack HK4. Moreover, as expected from the TCGA data, these liver cancer cell lines fall into two subpopulations: HK1−HK2+ (Hep3B, HepG2, JHH5, JHH7, Huh7) and HK1+HK2+ (HLF, JHH6) (Fig. 1e). We used these liver cancer cell lines to examine the therapeutic efficacy of the targeted inhibition of HK2.
HK2 shRNA-silencing mediated inhibition of proliferation and colony formation in HK1HK2cancer cells, but not HK1HK2cancer cells, is a property of cancer cells from a variety of tissues of origin − + + +
The lack of an shHK2 inhibitory response for HK1+HK2+ cancer cells is not unique to liver cancer cells; shHK2 inhibition of HK2 expression is unable to prevent proliferation or colony formation in HK1+HK2+ tumor cell lines derived from lung, breast, or colon cancers (Fig. 2c, d) as well as HK1+HK2+ liver cancer cells. In addition, we used CRISPR Cas9 HK1 deletion to create isogenic HK1−HK2+ and HK1+HK2+ H460 lung cancer cell lines (Additional file 1: Figure S1C). DOX-induced shHK2 expression in the HK1−HK2+ H460 cells suppressed colony formation but had no effect on isogenic HK1+HK2+ H460 cell colony formation (Fig. 2e); conversion of an HK1+HK2+ cancer cell to an isogenic HK1−HK2+ cancer cell has no effect on cell growth or proliferation but converts cells resistant to shHK2-mediated inhibition of proliferation or colony formation to cells sensitive to shHK2 inhibition.
Both HK1 and HK2 drive cancer aerobic glycolysis—glucose consumption and lactic acid production. Consequently, HK2 knockdown by DOX-induced shHK2 resulted in a greater reduction of both glucose consumption and lactate production more pronounced in HK1−HK2+ cancer cells (Hep3B, Huh7) compared to HK1+HK2+ cancer cells (SUM159) (Fig. 2f). However, before proceeding further with consideration of HK2-targeted therapy, we wanted to determine if reduction of HK2 activity in HK1−HK2+ cells would also reduce glucose consumption in vivo. In mouse subcutaneous Hep3B/shHK2DOX tumor xenografts, HK2 knockdown by a 4-day DOX treatment in the diet reduced tumor glucose consumption as determined non-invasively by 18F-fluorodeoxyglucose (FDG)/positron emission tomography (PET) imaging [18, 19] (Fig. 2g, h).
While HK2 knockdown in HK1−HK2+ liver cancer cells substantially reduced cell proliferation, the majority of the cells remained alive (Additional file 1: Figure S1D); after removing DOX from cultured Hep3B/shHK2DOX cells, the DOX-treated HK1−HK2+ cells were able to recover from HK2-inhibited cytostasis and resumed proliferation (Fig. 2i). These data suggest that monotherapy with selective HK2 inhibitors in HK1−HK2+ tumors is unlikely to be curative, either in preclinical models or in clinical applications; identification of synergistic or synthetically lethal agents in combination with HK2 inhibition will be necessary for HK2-targeted therapy of HK1−HK2+ tumors.
![HK1HK2liver cancer cells are sensitive to HK2 knockdown-induced growth inhibition.HK1HK2liver cancer cells are sensitive to HK2 knockdown-induced growth inhibition. Top panel, HK2 knockdown by DOX-inducible shHK2after 72 h DOX (25 ng/ml) induction. Bottom panel, after a 7-day exposure to DOX to induce shRNA expression, relative cell numbers were measured using the MTT assay. Data are expressed as “growth inhibition” [100 × (1 − MTT assay values of DOX-treated cells/MTT assay values of untreated cells)].Effect of DOX-induced HK2 knockdown on colony formation by HK1HK2and HK1HK2liver cancer cells. Triplicate wells of each cell line are shown, stained at 15–20 days after plating.,DOX-induced HK2 silencing has limited or no effect oncell proliferation orcolony formation in HK1HK2cancer cells, regardless of tissue of origin.DOX-induced shRNA HK2 knockdown inhibited colony formation by H460HK1/shHK2isogenic cells but not by isogenic H460/shHK2cells.HK2 knockdown inhibits glucose consumption (upper panels) and lactate production (lower panels) in Hep3B/shHK2, Huh7/shHK2, and SUM159/shHK2cells. Triplicate wells of cells were exposed to DOX or vehicle for 72 hwere then refreshed and, after 24 h, glucose consumption and lactate production were measured.HK2 knockdown decreasesF-FDG/PET signal in a mouse subcutaneous Hep3B/shHK2xenograft model. Tumors on the left flank: Hep3B/shCtrl. Tumors on the right flank: Hep3B/shHK2.F-FDG PET/CT scans were performed for each mouse before and after mice were switched to a DOX diet for 4 days.Following the PET scans in panel, HK2 knockdown was confirmed in tumor extracts by Western blotting.Cytostasis induced in HK1HK2liver cancer cells by HK2 knockdown is reversible. Hep3B/shHK2cells and Hep3B/shCtrlcells were treated with DOX for 2 days then re-plated in equal numbers in 96-well plates on day 0 in the continued presence of DOX. Cell proliferation during days 0–5 in the presence of DOX was measured (left panel). Cells from the two conditions (+DOX, −DOX) were pooled and cultured in DOX-free medium for 7 days (days 5–12) to allow the degradation of shRNAs in the cells and re-expression of HK2. The cells were then re-plated in 96-well plates and grown in the absence of DOX to generate the growth curves for days 12–16 (right panel). Relative cell proliferation was measured by the MTT assay and presented as fold change compared to the value on day 0. Each data point in panels,, andrepresents mean ± SD of triplicate samples − + − + DOX − + + + + + KO DOX DOX DOX DOX DOX 18 DOX DOX DOX 18 − + DOX DOX a b c d c d e f g h g i a c f . Media](https://europepmc.org/articles/PMC6022704/bin/40170_2018_181_Fig2_HTML.jpg.jpg "Click to view full size")
HK1HK2liver cancer cells are sensitive to HK2 knockdown-induced growth inhibition.HK1HK2liver cancer cells are sensitive to HK2 knockdown-induced growth inhibition. Top panel, HK2 knockdown by DOX-inducible shHK2after 72 h DOX (25 ng/ml) induction. Bottom panel, after a 7-day exposure to DOX to induce shRNA expression, relative cell numbers were measured using the MTT assay. Data are expressed as “growth inhibition” [100 × (1 − MTT assay values of DOX-treated cells/MTT assay values of untreated cells)].Effect of DOX-induced HK2 knockdown on colony formation by HK1HK2and HK1HK2liver cancer cells. Triplicate wells of each cell line are shown, stained at 15–20 days after plating.,DOX-induced HK2 silencing has limited or no effect oncell proliferation orcolony formation in HK1HK2cancer cells, regardless of tissue of origin.DOX-induced shRNA HK2 knockdown inhibited colony formation by H460HK1/shHK2isogenic cells but not by isogenic H460/shHK2cells.HK2 knockdown inhibits glucose consumption (upper panels) and lactate production (lower panels) in Hep3B/shHK2, Huh7/shHK2, and SUM159/shHK2cells. Triplicate wells of cells were exposed to DOX or vehicle for 72 hwere then refreshed and, after 24 h, glucose consumption and lactate production were measured.HK2 knockdown decreasesF-FDG/PET signal in a mouse subcutaneous Hep3B/shHK2xenograft model. Tumors on the left flank: Hep3B/shCtrl. Tumors on the right flank: Hep3B/shHK2.F-FDG PET/CT scans were performed for each mouse before and after mice were switched to a DOX diet for 4 days.Following the PET scans in panel, HK2 knockdown was confirmed in tumor extracts by Western blotting.Cytostasis induced in HK1HK2liver cancer cells by HK2 knockdown is reversible. Hep3B/shHK2cells and Hep3B/shCtrlcells were treated with DOX for 2 days then re-plated in equal numbers in 96-well plates on day 0 in the continued presence of DOX. Cell proliferation during days 0–5 in the presence of DOX was measured (left panel). Cells from the two conditions (+DOX, −DOX) were pooled and cultured in DOX-free medium for 7 days (days 5–12) to allow the degradation of shRNAs in the cells and re-expression of HK2. The cells were then re-plated in 96-well plates and grown in the absence of DOX to generate the growth curves for days 12–16 (right panel). Relative cell proliferation was measured by the MTT assay and presented as fold change compared to the value on day 0. Each data point in panels,, andrepresents mean ± SD of triplicate samples − + − + DOX − + + + + + KO DOX DOX DOX DOX DOX 18 DOX DOX DOX 18 − + DOX DOX a b c d c d e f g h g i a c f . Media
Diphenyleneiodonium (DPI), in combination with HK2 knockdown, is synthetically lethal for HK1HK2liver cancer cells − +
DPI has been reported to inhibit NADPH oxidase (NOX) [22], nitric oxide synthase (NOS) [23], and mitochondrial complex I [24]. Tested in combination with HK2 knockdown, neither NOX inhibitors (GKT137831, apocynin; Additional file 1: Figure S3A) nor NOS inhibitors (L-NAME or L-NNA) (Additional file 1: Figure S3B) show synergy in HK1−HK2+ liver cancer cells. In contrast, the complex I inhibitor rotenone (ROT) is synthetically lethal in combination with HK2 knockdown (Additional file 1: Figure S3C). In addition, DPI treatment reduced, but did not eliminate, mitochondrial oxygen consumption (Fig. 3d), consistent with its inhibition of mitochondrial complex I, and increased cellular demand for glycolysis (Fig. 3e). These results indicate that DPI synergizes with HK2 silencing/inhibition in HK1−HK2+ liver cancer cells through the inhibition of mitochondrial complex I, reducing ATP production from the electron transport chain (ETC). The synergistic reduction in cellular energy levels by FDG/DPI or FDG/ROT triggered the activation of the cellular energy sensor AMPKα (Fig. 3f and Additional file 1: Figure S3D), suppressed the energy-dependent mTOR pathway (Fig. 3g and Additional file 1: Figure S3E) [25], and induced apoptosis (Fig. 3h and Additional file 1: Figure S3F).
In a mouse xenograft model of HK1−HK2+ Hep3B/shHK2DOX tumor progression, DOX-induced HK2 knockdown initiated when the tumors reached 200 mm3 significantly reduced tumor growth (Fig. 3i). Although DPI alone had no significant effect on tumor growth, the DPI/shHK2 combination was significantly more effective in reducing tumor growth when compared to the effect of either shHK2 or DPI alone (Fig. 3i). The Hep3B/shHK2DOX tumors, in which HK2 was reduced to undetectable levels, did not express other active HK isoforms to compensate for glycolysis (Additional file 1: Figure S4). The combination shHK2/DPI treatment resulted in increased levels of phosphorylated AMPKα and dephosphorylation of S6 and 4EBP1 in xenograft Hep3B/shHK2DOX tumors (Fig. 3j), demonstrating the reduction in tumor energy production induced by the shHK2/DPI combination therapy.
DPI is a synthetically lethal partner in combination with HK2 knockdown or inhibition in HK1HK2liver cancer cells.A high-throughput screen of 3205 drug-like compounds and 119 FDA-approved oncology drugs identified DPI as the best synergistic agent with HK2 knockdown in Hep3B/shHK2cells. Primary screening of all compounds at 10 μM was performed in combination with DOX-induced HK2 knockdown in Hep3B/shHK2cells. Growth inhibition was measured by the CellTiter-Glo assay (left panel). Ninety-two compounds withscores ≤ − 3 were tested in dose response curves (dose response concentrations 0, 0.0006, 0.0025, 0.01, 0.039, 0.156, 0.625, and 2.5 μM) in Hep3B/shHK2cells cultured with or without DOX. Viable cells were normalized to DMSO-treated cells with or without DOX, respectively (right panel). The dots represent compounds at the tested concentrations. The red line connects the individual DPI concentration points. The chemical structure of DPI is shown.DPI is synthetically lethal with HK2 knockdown or inhibition in HK1HK2liver cancer cells. DOX/DPI 2-day DOX pretreatment prior to 72 h DPI (100 nM) treatment. FDG/DPI 72 h FDG (1 mM) and/or DPI (100 nM) treatments. Viability percentages were determined by trypan blue staining.FDG/DPI combination irreversibly inhibits Hep3b and Huh7 HK1HK2liver cancer cell proliferation. Viable cells were determined by trypan blue staining.DPI decreases the oxygen consumption rate (OCR) of Hep3B cells. Cells were exposed to 100 nM DPI or vehicle for 2 h before OCR measurements.DPI increases glycolysis of HK1HK2liver cancer cells. Cells were pretreated with vehicle or DOX for 72 h prior to exposure to DPI (100 nM). Twenty-four hours later, culture media were analyzed for glucose and lactate.,FDG/DPI treatment activates AMPKα () and inactivates the mTOR pathway () in Hep3B cells. Cells were treated with 1 mM FDG and/or 100 nM DPI for 4 h.FDG/DPI treatment triggers apoptosis. Hep3B HK1HK2cells were treated with FDG (1 mM) and/or DPI (100 nM) for 24 h prior to apoptosis analyses.DPI (2 mg/kg) enhances inhibition of tumor progression in response to HK2 silencing in Hep3B/shHK2tumors. Mice-bearing tumor xenografts were treated when tumors reached 200 mm(day 0).Effects of HK2 knockdown and/or DPI (2 mg/kg) treatment on HK2 expression, AMPKα activation, and mTOR pathway inactivation in the xenograft Hep3B/shHK2tumors described in panel. Each data point in–andrepresents mean ± SD of triplicate samples, and each data point inandrepresents mean ± SEM of five samples. *< 0.05, ***< 0.001 − + DOX DOX DOX − + − + − + − + DOX 3 DOX a b c d e f g f g h i j i a c e d i z P P
Inhibition of fatty acid oxidation sensitizes HK1HK2liver cancer cells to the FDG/DPI combination − +
Perhexiline (PER), a FAO inhibitor in clinical use as an anti-angina drug in Australia and Asia and currently in clinical trials in the USA [13], sensitized HK1−HK2+ Hep3B liver cancer cells to the FDG/DPI combination (Fig. 4c). In the presence of FDG and DPI, at sub-optimal concentrations (lower than the concentrations used in previous experiments) that did not cause significant cell death either alone or in combination, the addition of PER to HK1−HK2+ liver cancer cells resulted in substantial lethality (Fig. 4d). A similar sensitizing effect was observed with another FAO inhibitor, etomoxir (Additional file 1: Figure S5B). While the FDG/DPI combination significantly decreased lipid droplets (lipid droplets are an indicator of cellular fatty acid abundance) in HK1−HK2+ liver cancer cells, PER addition prevented the disappearance of lipid droplets (Fig. 4e). In addition, PER synergized with the FDG/DPI combination to activate AMPKα, suppress the mTOR pathway, and trigger apoptosis (Fig. 4f). In contrast, there was no significant synthetic lethality induced by the triple combination of shHK2/DPI/PER in HK1+HK2+ cancer cells (Additional file 1: Figure S5C), suggesting that our shHK2/DPI/PER triple-combination treatment is selectively cytotoxic for HK1−HK2+ cancer cells and is likely to be tolerated by normal tissues. Taken together, our results indicate that FAO inhibition further sensitizes HK1−HK2+ cells to the combined inhibition of glycolysis and the mitochondrial electron transport chain (ETC) and that the triple combination of HK2 inhibition, DPI, and PER simultaneously target the components of multiple energy generation sources (glycolysis, OXPHOS, FAO) in HK1−HK2+ liver cancer cells, resulting in synthetic lethality in cells that express HK2 but not HK1.
![Inhibition of fatty acid oxidation sensitizes HK1HK2liver cancer cells to the cytotoxicity of the HK2 inhibition/DPI combination.FDG/DPI or FDG/ROT combinations induce ACC phosphorylation. HK1HK2liver cancer cells were treated with the indicated drug(s) for 24 h prior to analysis of ACC phosphorylation. FDG, 1 mM. DPI, 100 nM. ROT, 100 nM.Clinical liver cancer samples show upregulated expression of genes involved in fatty acid elongation and downregulated expression of genes involved in FAO compared to normal liver tissues. Data were obtained from Oncomine ().PER sensitizes Hep3B cells to FDG/DPI treatment. Cells were treated with FDG, plus or minus 5 μM PER, for 72 h and then assayed for viable cells by the MTT assay. Open surface, [DPI + FDG + vehicle] treatment. Filled surface, [DPI + FDG + PER] treatment.PER sensitizes HK1HK2liver cancer cells to the cytotoxicity of FDG/DPI treatment. Triplicate wells of (left) Hep3B or (right) Huh7 cells were treated with FDG (250 μM), DPI (15 nM), and/or PER (5 μM) for 72 h, followed by trypan blue staining to determine cell viability. Viability % = 100% × (trypan blue negative cells/total cells). Data are means ± SD.PER rescues FDG/DPI-induced lipid droplet depletion in Hep3B cells. Hep3B cells were treated with DMSO, FDG (1 mM)/DPI (100 nM), or FDG (1 mM)/DPI (100 nM)/PER (5 μM) for 24 h, followed by Oil Red staining of intracellular lipid droplets. Representative images with indicated treatments are shown. Scale bars, 30 μM.FDG, DPI, and/or PER modulation of AMPKα activation, mTOR pathway inactivation, and apoptosis induction in Hep3B/shHK2cells. Cells were treated with FDG (250 μM), DPI (15 nM), and/or PER (5 μM) for 24 h, and cell extracts were examined by Western blotting − + − + − + DOX a b c d e f http://www.oncomine.com](https://europepmc.org/articles/PMC6022704/bin/40170_2018_181_Fig4_HTML.jpg.jpg "Click to view full size")
Inhibition of fatty acid oxidation sensitizes HK1HK2liver cancer cells to the cytotoxicity of the HK2 inhibition/DPI combination.FDG/DPI or FDG/ROT combinations induce ACC phosphorylation. HK1HK2liver cancer cells were treated with the indicated drug(s) for 24 h prior to analysis of ACC phosphorylation. FDG, 1 mM. DPI, 100 nM. ROT, 100 nM.Clinical liver cancer samples show upregulated expression of genes involved in fatty acid elongation and downregulated expression of genes involved in FAO compared to normal liver tissues. Data were obtained from Oncomine ().PER sensitizes Hep3B cells to FDG/DPI treatment. Cells were treated with FDG, plus or minus 5 μM PER, for 72 h and then assayed for viable cells by the MTT assay. Open surface, [DPI + FDG + vehicle] treatment. Filled surface, [DPI + FDG + PER] treatment.PER sensitizes HK1HK2liver cancer cells to the cytotoxicity of FDG/DPI treatment. Triplicate wells of (left) Hep3B or (right) Huh7 cells were treated with FDG (250 μM), DPI (15 nM), and/or PER (5 μM) for 72 h, followed by trypan blue staining to determine cell viability. Viability % = 100% × (trypan blue negative cells/total cells). Data are means ± SD.PER rescues FDG/DPI-induced lipid droplet depletion in Hep3B cells. Hep3B cells were treated with DMSO, FDG (1 mM)/DPI (100 nM), or FDG (1 mM)/DPI (100 nM)/PER (5 μM) for 24 h, followed by Oil Red staining of intracellular lipid droplets. Representative images with indicated treatments are shown. Scale bars, 30 μM.FDG, DPI, and/or PER modulation of AMPKα activation, mTOR pathway inactivation, and apoptosis induction in Hep3B/shHK2cells. Cells were treated with FDG (250 μM), DPI (15 nM), and/or PER (5 μM) for 24 h, and cell extracts were examined by Western blotting − + − + − + DOX a b c d e f http://www.oncomine.com
The HK2i/DPI/PER combination suppresses energy generation and alters cell metabolism in HK1HK2liver cancer cells − +
Mass spectrometry was used to determine the levels of energy-related molecules extracted from HK1−HK2+ Hep3B cells after an 8-h treatment with FDG, DPI, and/or PER at sub-optimal concentrations, at which each individual agent did not substantially affect cell growth (Fig. 4c, d) or OCR (Fig. 5a). The increase in the ADP/ATP and AMP/ATP ratios in response to the triple treatment demonstrated the combined inhibition of ATP production from glycolysis, oxidative phosphorylation, and FAO (Fig. 5c). In addition, the combination of PER with FDG, DPI, or FDG/DPI substantially increased the creatine/P-creatine ratio (Fig. 5c), limiting the ability of HK1−HK2+ liver cancer cells to replenish ATP from P-creatine. These molecular energy changes, measured after an 8-h treatment, occurred before cell death as measured by trypan blue exclusion (Additional file 1: Figure S6A). The FDG/DPI/PER-induced reduction in ATP was also confirmed in living Hep3B cells expressing firefly luciferase, an ATP-dependent reporter enzyme (Additional file 1: Figure S6B). Metabolomic analysis also revealed that the pools of most metabolites of glycolysis and the TCA cycle, as well as nucleotide pools, were reduced in HK1−HK2+ liver cancer cells treated with FDG/DPI and were further decreased when PER was added into the combination (Fig. 5d). These data suggest that global changes in cell metabolism, in association with or caused by energy inhibition, may contribute to the HK2i/DPI/PER-induced synthetic lethality in HK1−HK2+ cancer cells.
Modulation of energy generation and metabolism in HK1HK2Hep3B/shHK2cells in response to FDG/DPI/PER treatment.FDG/DPI/PER combination treatment acutely decreases the OCR of Hep3B cells. FDG (250 μM), DPI (15 nM), and/or PER (5 μM) were added to cultured Hep3B cells, as single agents or in the combinations shown, at time 0. The OCR was measured by the Seahorse assay. Oligomycin (2 μM), FCCP (0.1 μM), and Antimycin (2 μM) were used to indicate the fraction of respiration coupled to oxidative ATP production, maximum mitochondrial respiration, and non-mitochondrial respiration, respectively.OCR in Hep3B cells after 3, 8, and 24 h of drug exposure; concentrations are as in panel.The HK2i/DPI/PER triple combination decreases liver cancer cellular energy levels. After an 8-h drug treatment, AMP, ADP, ATP, creatine, and P-creatine amounts in Hep3B cells were determined by LC-MS.Changes in pool sizes of glycolysis and TCA cycle metabolites, as well as purine and pyrimidine nucleotides, after vehicle, FDG/DPI, or FDG/DPI/PER treatment in Hep3B cells. F1,6BP fructose 1,6-biphosphate, G3P glyceraldehyde 3-phosphate, DHAP dihydroxyacetone phosphate, 3PG 3-phospho-glycerate, PEP phosphoenolpyruvate, Cit citrate, α-KG α-ketoglutarate, Succ succinate, Fum fumarate, Mal malate. Each data point inandrepresents mean ± SEM of five samples, and each data point inandrepresents mean ± SD of triplicate samples. *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001 − + DOX a b a c d a b c d P P P P
The shHK2/DPI/PER combination suppresses established HK1HK2liver tumor progression in vivo − +
The potency of the shHK2/DPI/PER combination was also examined with two additional HK1−HK2+ liver tumor xenografts, Huh7/shHK2DOX and HepG2/shHK2DOX (Fig. 6e–g). These results confirm the in vivo efficacy and safety of our triple-combination therapy in established HK1−HK2+ liver tumors.
![PER sensitizes established xenograft HK1HK2liver tumors to HK2 knockdown/DPI combination therapy.PER enhances the ability of the HK2 knockdown/DPI combination to retard the progression of Hep3B/shHK2tumor xenografts. When tumors reached 200 mm(day 0), xenografts were randomized into a control group and two treatment groups. The control mice (= 7) were remained on the standard diet and were treated with vehicle. In the treatment groups, mice were placed on a DOX-supplemented diet (from day 0) and treated either with DPI (2 mg/kg, daily i.p. from day 3,= 7) or with [DPI (2 mg/kg, daily i.p.) + PER (30 mg/kg, daily i.p), from day 3,= 8].Representative images of tumor progression in three mice from each group in panel.Body weights of the mice in panelbearing xenograft subcutaneous tumors, in response to the indicated treatments.shHK2/DPI/PER treatment activates AMPKα and dephosphorylates S6 and elicits cleavage of caspase-3 and caspase-7. Hep3B/shHK2tumors from the indicated treatment groups were collected at day 15. Protein extracts from tissue homogenate supernatants were analyzed.PER enhances the ability of the HK2 knockdown/DPI combination to retard the progression of HK1HK2Huh7shHK2liver tumor xenografts. Experimental conditions are the same as those described in panel(= 6).PER enhances the ability of the HK2 knockdown/DPI combination to retard the progression of HK1HK2HepG2/shHK2liver tumor xenografts. Experimental conditions are the same as those described in panel(= 5).HK2 knockdown/DPI/PER combination suppresses HepG2/shHK2tumor growth. Weights of HepG2/shHK2tumors after indicated treatments of the xenograft-bearing mice shown in panelare shown. All data are expressed as means ± SEM. *< 0.05. **< 0.01. ***< 0.001 − + DOX 3 DOX − + DOX − + DOX DOX DOX a b a c a d e a f a g f n n n n n P P P](https://europepmc.org/articles/PMC6022704/bin/40170_2018_181_Fig6_HTML.jpg.jpg "Click to view full size")
PER sensitizes established xenograft HK1HK2liver tumors to HK2 knockdown/DPI combination therapy.PER enhances the ability of the HK2 knockdown/DPI combination to retard the progression of Hep3B/shHK2tumor xenografts. When tumors reached 200 mm(day 0), xenografts were randomized into a control group and two treatment groups. The control mice (= 7) were remained on the standard diet and were treated with vehicle. In the treatment groups, mice were placed on a DOX-supplemented diet (from day 0) and treated either with DPI (2 mg/kg, daily i.p. from day 3,= 7) or with [DPI (2 mg/kg, daily i.p.) + PER (30 mg/kg, daily i.p), from day 3,= 8].Representative images of tumor progression in three mice from each group in panel.Body weights of the mice in panelbearing xenograft subcutaneous tumors, in response to the indicated treatments.shHK2/DPI/PER treatment activates AMPKα and dephosphorylates S6 and elicits cleavage of caspase-3 and caspase-7. Hep3B/shHK2tumors from the indicated treatment groups were collected at day 15. Protein extracts from tissue homogenate supernatants were analyzed.PER enhances the ability of the HK2 knockdown/DPI combination to retard the progression of HK1HK2Huh7shHK2liver tumor xenografts. Experimental conditions are the same as those described in panel(= 6).PER enhances the ability of the HK2 knockdown/DPI combination to retard the progression of HK1HK2HepG2/shHK2liver tumor xenografts. Experimental conditions are the same as those described in panel(= 5).HK2 knockdown/DPI/PER combination suppresses HepG2/shHK2tumor growth. Weights of HepG2/shHK2tumors after indicated treatments of the xenograft-bearing mice shown in panelare shown. All data are expressed as means ± SEM. *< 0.05. **< 0.01. ***< 0.001 − + DOX 3 DOX − + DOX − + DOX DOX DOX a b a c a d e a f a g f n n n n n P P P
The shHK2/DPI/PER combination is an effective precision therapy for treating HK1HK2tumors, regardless of their tissues of origin − +
The shHK2/DPI/PER combination does not inhibit proliferation or tumor progression for HK1HK2cancer cells but is effective in inhibiting proliferation and progression for the isogenic HK1HK2cancer cells.Effect of the shHK2/DPI/PER combination on isogenic HK1HK2H460WT/shHK2and H460HK1/shHK2cell proliferation. Cells were pretreated with vehicle or DOX for 2 days, followed by a 72-h treatment with DPI in the indicated concentration range, in the presence or absence of DOX and the presence or absence of PER (5 μM). Upper panels, MTT assay values were normalized to control samples (-DOX, -DPI, -PER) of the individual isogenic cell lines. Data are expressed as means ± SD. Lower panels, representative images of MTT assay results.The shHK2/DPI/PER combination triggers apoptosis in H460HK1/shHK2cells but not in H460WT/shHK2cells. Cells were pretreated with vehicle or DOX for 2 days, followed by a 24-h treatment with 15 nM DPI, 5 μM PER, and DOX, both as single agents and in combination (Comb). Cell lysates were examined by Western blot for HK1, HK2, and cleaved caspase 3 expression.The shHK2/DPI/PER combination inhibits the progression of H460HK1/shHK2tumor xenografts but not H460WT/shHK2tumor xenografts. When tumors reached 200 mm(day 0), xenografts were randomized into five groups (= 5 per group) receiving vehicle, DOX in the diet, DPI (2 mg/kg, daily i.p.), and PER (30 mg/kg, daily i.p.), both as single agents and in combination. Data are expressed as means ± SEM. **< 0.01. ***< 0.001. NS not significant.The shHK2/DPI/PER combination triggers apoptosis in H460HK1/shHK2tumor xenografts but not H460WT/shHK2tumor xenografts. Three representative xenograft tumors in each group in panelwere homogenized for Western blotting analyses of indicated proteins + + KO + + + DOX KO DOX KO DOX DOX KO DOX DOX 3 KO DOX DOX a b c d c n P P
Discussion
In this study, we describe a triple-combination therapy for treatment of HK1−HK2+ cancers that targets the HK1−HK2+ cancer-specific modes of energy generation, HK2-driven glycolysis, mitochondrial oxidative phosphorylation, and fatty acid oxidation. Using HK1−HK2+ liver cancer cells as an example, we illustrated that targeting each individual energy generation source is either cytostatic or has only a subtle effect on cell proliferation or tumor progression for HK1−HK2+ liver cancer cells. While the majority of cancers including liver cancer, from many tissues of origin, express both HK1 and HK2, in many types of cancers, there exist subsets of HK1−HK2+ tumors, i.e., tumors that express only HK2. The potential specificity of this therapy for this HK1−HK2+ subclass, and the extension of this therapy across cancers from a broad range of tissues of origin, results from this common phenotype that makes them vulnerable to this combination therapy.
We have studied HK2 knockdown in a large panel of naturally existing HK1+HK2+ and HK1−HK2+ cancer cells and found that only HK1−HK2+ cancer cells but not HK1+HK2+ cells as defined by mRNA and Western blot analyses are sensitive to HK2 knockdown. It is likely that there is a threshold effect for HK1 expression necessary to provide resistance to HK2 inhibition. Future studies in the titration of HK1 protein levels using genetic manipulation approaches, such as inducible knockdown of HK1 in HK1+HK2+ cancer cells with different concentrations of doxycycline or inducible expression of HK1 in HK1−HK2+ cancer cells with different concentrations of doxycycline, would quantify the threshold effect of HK1 required to provide resistance to HK2 inhibition in cancer cells. In addition, how the HK1 gene is silenced in the HK1−HK2+ subsets of cancers is unknown. Future studies in regulation of HK1 expression in cancers and in normal tissues may provide targets for combination therapies in conjunction with HK2 inhibition for cancer treatment.
It is important to note that, even at the highest DPI concentration (100 nM) we used in cell culture experiments, which is relevant to the tolerated plasma concentration previously determined in mice [28], DPI inhibits only 50% of OXPHOS activity (Fig. 3d). These data suggest that the residual OXPHOS system can still oxidize fuels provided by metabolic pathways such as pyruvate oxidation, glutamine catabolism, and FAO. When we partially inhibited HK2-driven glycolysis and OXPHOS in HK1−HK2+ cancer cells, we observed that FAO was upregulated as indicated by ACC phosphorylation (Fig. 4a) and disappearance of intracellular lipid droplets (Fig. 4e). Therefore, we targeted FAO with the clinical FAO inhibitor PER. The addition of PER sensitized HK1−HK2+ cancer cells to the combination of FDG and DPI, indicating that FAO plays an important role in providing fuels to the residual OXPHOS activity, among the different fuel-providing pathways.
Because cancer cells can flexibly reprogram their energy generation [29], loss of HK2 activity in HK1−HK2+ cancer cells is only cytostatic; however, the combined inhibition of these three critical pathways involved in ATP production leads to a profound decrease in energy generation and to synthetic lethality for HK1−HK2+ cancers. Identification/stratification of patients with an HK1−HK2+ cancer molecular characteristic will be essential for clinical translation of our combination therapy as a precision medicine for this tumor subtype in tumors of different origins.
While our manuscript was in preparation for submission, De Waal et al. [11] reported that cell proliferation and xenograft progression of HepG2 and Huh7 liver cancer cells, which are from the HK1−HK2+ liver cancer subset we studied (Fig. 1e), are restricted by shHK2DOX expression and that combination of shHK2DOX expression and OXPHOS inhibition with metformin further suppressed Huh7 xenograft progression. We also find shHK2DOX HK2 inhibition plus OXPHOS inhibition (in our case with DPI) reduces, but does not completely suppress, xenograft tumor progression for Huh7 (Fig. 6e) and Hep3B (Fig. 6a) HK1−HK2+ liver cancer xenografts. We suggest inhibition of the third major pathway driving ATP production, fatty acid oxidation, would enhance inhibition (by shHK2DOX + DPI/metformin) of HK1−HK2+ liver tumor xenograft growth and confirm this hypothesis for Hep3B, Huh7, and HepG2 liver tumor xenograft growth (Fig. 6a, e, and f).
De Waal et al. [11] emphasized that many liver cancers express HK2; however, a substantial proportion of liver cancers express both HK1 and HK2 or only HK1 (Fig. 1c). We find neither cell proliferation (Fig. 2a) nor colony formation (Fig. 2b) are affected by shHK2DOX HK2 inhibition for HK1+HK2+ HLF and JHH6 liver cancer cells, suggesting pharmacologic HK2 inhibition will not have therapeutic efficacy for HK1+HK2+ liver cancers.
CCLE cell gene expression profile analyses revealed cancers from tissues of multiple origins contain HK1−HK2+ subsets (Fig. 1d). To determine whether HK1−HK2+ cancers of other origins will be sensitive to HK2 inhibition, we created isogenic HK1−HK2+ cells from HK1+HK2+ H460 lung cancer cells. As expected, xenograft progression of HK1+HK2+ H460 cells was unaffected by shHK2DOX expression or the shHK2DOX/DPI/PER combination (Fig. 7c). In contrast, HK1KOHK2+ H460 xenograft progression was inhibited by shHK2DOX expression or shHK2DOX/DPI/PER (Fig. 7c). We conclude (i) that inhibition of HK2-driven glycolysis, OXPHOS, and FAO is likely to be a pan-tumor precision therapy approach to HK1−HK2+ tumors, regardless of their tissue of origin, and (ii) that cancer therapies that involve HK2 inhibition will be restricted to tumors that do not express HK1.
In addition to the hexokinases, several isoform switches in other metabolic enzymes occur during hepatocarcinogenesis, e.g., a splicing switch from ketohexokinase-C (KHK-C) to KHK-A involved in fructose metabolism [30], and a switch from 11β-HSD1 to 11β-HSD2 involved in glucocorticoid metabolism and gluconeogenesis [31]. Although the mechanisms of these metabolic isozyme switches and possible relationships among these switches during hepatocarcinogenesis are not currently understood, these additional cancer-specific isoforms may also be therapeutic targets for the development of effective cancer treatments.
Tumor progression for HK1−HK2+ liver cancer cells either by shHK2DOX + metformin [11] or by shHK2DOX + DPI (Fig. 3) is retarded but not completely suppressed. In vivo, DPI is maximally tolerated by mice at 2 mg/kg, with a plasma Cmax value of 1 μM [28]. The combination of shRNA HK2 knockdown and daily DPI i.p. injection at 2 mg/kg did not cause HK1−HK2+ liver tumor cell apoptosis (Fig. 6d), suggesting that DPI at this dosage, in combination with HK2 knockdown, is not sufficient to cause lethality of HK1−HK2+ tumor cells in vivo. However, including PER in vivo to further sensitize HK1−HK2+ cancer cells to the shHK2/DPI combination improved the therapeutic effect on HK1−HK2+ tumors from growth inhibition to tumor cell death. Targeting fatty acid oxidation has recently been attracting additional attention for cancer therapy [13, 32, 33].
ATP is the principal energy currency in cell metabolism and is a versatile regulator of cellular activities [34, 35]. Maintenance of an adequate ATP supply is of crucial importance for cellular functions, for both normal tissues and for cancers. Targeting HK2-mediated glycolysis, mitochondrial respiration, and fatty acid oxidation in liver HK1−HK2+ cancer cells results in a synergistic decrease in cancer energy/ATP production. While a substantial drop in cellular energy levels causes HK1−HK2+ cancer cell death, the inhibition of different ATP generation sources may also have a profound impact on the distribution of compartmentalized intracellular ATP concentrations (“ATP inhomogeneity”) [36]. For example, ATP produced from glycolysis is readily diffused in the cytosol, whereas transfer of respiration-derived ATP from mitochondrial to cytosol requires carrier proteins across mitochondrial membranes [37]. Nuclear ATP levels are compromised by inhibiting mitochondrial ATP production but not by inhibiting glycolysis [38]. The subcellular compartmentalization of ATP has important roles in multiple cellular activities, such as cell motility [36], muscle contraction [39], DNA repair [40], and chromatin remodeling [38]. Further study will be required to explore the impact of our combination therapy on the ATP inhomogeneity in HK1−HK2+ cancer cells derived from alternative tissues and the consequent effects on cell viability.
To translate our findings to clinical treatment for HK1−HK2+ cancers, several steps will be required: (1) shHK2DOX HK2 inhibition needs to be replaced by a selective HK2 inhibitor. GlaxoSmithKline recently reported their development of HK2-preferental inhibitors [10]. (2) DPI stood out among all screened compounds as the best synergistic partner with HK2 knockdown in HK1−HK2+ liver cancer cells. Although DPI has been extensively tested in pre-clinical animal models [28, 41–44], its safety in human subjects is not known. Clinical drugs with ETC-targeting effects, such as metformin [45] and papaverine [46], will need to be examined for their abilities to replace DPI in the combination therapy. (3) Doses, schedules, and routes of administration of the therapeutic agents involved in the triple combination will need optimization; the dosages in this study for xenograft tumor analyses were based on the previously reported single-agent dosages in mouse studies [28, 47]. Because of the synergistic effects, the required dosage of each agent for optimal combination therapy may well be lower than those used in these initial studies.
Conclusions
We identified the HK1−HK2+ cancer subsets existing among a wide variety of cancer types. HK2 is the only active HK isoform driving glycolysis in the HK1−HK2+ subsets of cancers. The combination therapy we have developed specifically reduced the cellular ATP level in these HK1−HK2+ cancer cells to a level incompatible with cell survival. In conclusion, we suggest that reduction of ATP levels to a level that will no longer support the survival of HK1−HK2+ cancer cells can be clinically exploited as a targeted, precision medicine for HK1−HK2+ tumors, regardless of their tissue of origin.
Additional files
Additional file 1:Figure S1. HK1−HK2+ cancer cells are highly sensitive to HK2 knockdown-induced growth inhibition. Figure S2. DPI synergizes with HK2 knockdown or inhibition in HK1−HK2+ liver cancer cells. Figure S3. DPI synergizes with HK2 silencing/inhibition by targeting mitochondrial complex I in HK1−HK2+ liver cancer cells. Figure S4. HK isoform expression in Hep3B/shHK2DOX xenograft tumors with DOX and/or DPI treatments. Figure S5. Inhibition of fatty acid oxidation sensitizes HK1−HK2+ liver cancer cells to the HK2 inhibition/DPI combination. Figure S6. Modulation of HK1−HK2+ liver cancer cellular metabolism by the HK2i/DPI/PER combination. Figure S7. PER as a single agent does not have a significantly detectable effect on growth of subcutaneous Hep3B/shHK2DOX tumors. (PPTX 782 kb)Additional file 2:Table S1. The list of 119 FDA-approved oncology drugs provided by the National Cancer Institute (NCI) tested for synergy with DOX treatment in Hep3B/shHK2DOX cells. Table S2. Synergy between HK2 inhibition and DPI. (DOCX 44 kb)